Title:
Evaluation of Adhesion Force and Binding Affinity of Phytohemagglutinin Erythroagglutinating to
EGF Receptor on Human Lung Cancer Cells
Running title:
Binding Affinity and Force of PHA-E to EGFR
Byline:
Wei-Ting Kuo, Guo-Chung Dong, Chun-Hsu Yao, Jian-Yuan Huang, Feng-Huei Lin
Correspondent footnote:
Feng-Huei Lin
Professor
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan
Fax: 886-2-23940049; Tel: 886-2-23912641; e-mail: double@ntu.edu.tw
Keywords:
Adhesion Force; Binding Affinity; Epidermal Growth Factor Receptor; Lung Cancer; Natural Product;
Phytohemagglutinin Erythroagglutinating; Target Molecule
1
Evaluation of Adhesion Force and Binding Affinity of Phytohemagglutinin Erythroagglutinating
to EGF Receptor on Human Lung Cancer Cells
Wei-Ting Kuo1, Guo-Chung Dong2, Chun-Hsu Yao3,4, Jian-Yuan Huang1, Feng-Huei Lin1,2*
1
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan
2
Division of Medical Engineering Research, National Health Research Institutes, Miaoli, Taiwan
3
Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung,
Taiwan
4
School of Chinese Medicine, China Medical University, Taichung, Taiwan
*Corresponding Author
Feng-Huei Lin
Professor
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan
Fax: 886-2-23940049; Tel: 886-2-23912641; e-mail: double@ntu.edu.tw
2
Abstract
PHA-E is a natural product extracted from red kidney beans, and it has been reported to induce cell
apoptosis by blocking EGFR in lung cancer cells. Because EGF is the major in vivo competitor to
PHA-E in clinical application, PHA-E must be proved that has better affinity to EGFR than EGF. This
study would focus on how PHA-E tightly bind to EGFR and the results would compare with EGF. The
adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that was higher
than EGF (183.65±86.93 pN). The equilibrium dissociation constant of PHA-E and EGF to EGFR was
2.4×10-91.4×10-9 and 7.3×10-82.7×10-8, respectively, that could evaluate binding affinity. The result
showed that binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the
results of flow cytometer and confocal microscope, we found binding efficiency of EGF to EGFR was
decrease as the concentration of PHA-E increased. In the analysis of Western blot, treatment of A-549
cells with PHA-E resulted in a dose-dependent decrease in EGFR phosphorylation. In conclusion, we
found that PHA-E had better adhesion force and binding affinity to EGFR than that of the EGF. The
interaction between PHA-E and EGFR could block EGF binding and then inhibit EGFR
phosphorylation. PHA-E could be developed into a new target molecule for lung cancer treatment that
could be immobilized on the drug carrier to guide therapeutic particles to the tumor site.
Keywords: Adhesion Force; Binding Affinity; Epidermal Growth Factor Receptor; Lung Cancer;
Natural Product; Phytohemagglutinin Erythroagglutinating; Target Molecule
3
1. Introduction
Lung cancer is the leading cause (23%) of cancer-related deaths in males and the second leading
cause (13%) of the total cancer deaths in females around the world [1]. It can be generally divided into
small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC accounts for
approximately 85% of lung cancer cases and the main histologic types are adenocarcinoma,
squamous-cell carcinoma, and large-cell carcinoma [2]. Epidermal growth factor receptor (EGFR) is
overexpressed in many epithelial cancers, including NSCLC, and occurs in 40-80% of patients with
NSCLC [3].
EGFR is a member of ErbB family of receptor tyrosine kinases (RTKs) [4], and it is a
transmembrane glycoprotein comprised of extracellular ligand-binding domain, transmembrane domain,
intracellular protein tyrosine kinase domain and C-terminal domain [5]. Activation of EGFR occurs
when a ligand such as epidermal growth factor (EGF) binds to the extracellular domain and induces
conformational changes that cause EGFR to be homodimer with itself or heterodimer with other
members [6], and then directly active tyrosine kinase domain and ensuing auto-phosphorylation of
C-terminal tyrosine residues, finally induces the downstream signaling pathway [7]. Over expression of
EGFR is associated with several hallmarks of cancer, including inhibition of apoptosis, sustained
angiogenesis, proliferation and survival, and tissue invasion and metastasis [8].
EGFR inhibitors have been investigated as first-line or subsequent therapy options for patients with
NSCLC [9]. There are two classes of EGFR inhibitors in clinical use in NSCLC. One is monoclonal
antibodies that block ligand binding and receptor activation by targeting the extracellular domain of
EGFR. The other is small-molecule tyrosine kinase inhibitors that inhibit the tyrosine kinase activity by
competing with ATP for ATP-binding site [10]. These EGFR inhibitors could stop cell growth and
induce apoptosis of cancer [11]. However, it would be time-consuming and very costly to produce
EGFR inhibitors from several steps by genetic engineering method [12]. In addition, some side effects
have been in many literatures such as acneiform eruption, xerosis cutis, nail abnormalities, folliculitis,
hair modifications, mucosal disorders, etc [13,14]. The drug-resistance was also reported after long
term administration [15,16]; that forced to consider the other anti-cancer drugs in cancer treatment.
Over the last two decades, scientists and researchers tried to look for the substances extracted from the
natural products or herbs [17]; that could effectively bind to EGFR as the substitute of EGFR inhibitors
to lower down the side effects and cost.
4
Phytohemagglutinin erythroagglutinating (PHA-E) is a phytohemagglutinin (PHA) isoform which
is derived from red kidney beans (Phaseolus vulgarus), and it is a glycoprotein of approximately 125
kDa. PHA is usually used for the activation of normal T cells and has been demonstrated to exhibit
antifungal, antiviral, and HIV-1 reverse transcriptase inhibitory activities [18]. PHA was also reported
to inhibit the growth of human cancer cells [19,20]. In the previous study, we found PHA-E could
induce growth inhibition and cytotoxicity of lung cancer cells, which was mediated through blocking
EGFR pathway and activation of the mitochondria apoptosis pathway [21]. However, EGF is the major
in vivo competitor to PHA-E in clinical application; PHA-E must be proved that has better affinity to
EGFR than EGF.
Atomic force microscope (AFM) and surface plasmon resonance (SPR) are powerful techniques for
analyzing biomolecular affinity [22]. AFM can be designed as a high resolution scanning machine [23],
and it is also a useful tool for the measurement of the adhesion forces between ligands and receptors
[24,25]. When the AFM tips are coated ligands and the substrates are immobilized receptors, the
process is required bringing the tip in contact with the surface so that to form a ligand-receptor
complex. And then the tip is retracted from the substrate surface, the rupture force for dissociation of
pulling away the tip from the substrate can be measured through the deflection of the cantilever which
is detected by a laser beam aimed at the cantilever and reflected onto a photodiode detector [26]. SPR
biosensor technology could be used to measure reaction kinetics and calculating affinity constants of
biomolecular interaction in real time with a high degree of sensitivity and without labeling
requirements [27,28]. When the activated surface of sensor chip is immobilized receptors, the ligands
in solution will flow through surface. Binding of interacting ligands to surface-immobilized receptors
alters the mass of surface layer. SPR detects change in the refractive index and the shift of resonant
angle of reflected light can be monitored in real time as a plot of resonance signal versus time [29,30].
This study would focus on how PHA-E tightly binds to EGFR and the results would compare with
EGF. If the PHA-E showed better affinity to EGFR, it could compete with EGF to bind to EGFR that
block the following cellular reactions as done by EGFR inhibitor; which should have a great potential
as one of anti-cancer drugs with low cost and side effects.
2. Materials and methods
2-1 Materials and reagents
5
AFM tips were purchased from Asylum Research (California, USA). Sensor CM5 chips were
obtained from Biacore Life Science (Uppsala, Sweden). EGF was purchased from PeproTech (New
Jersey, USA). EGFR and PHA-E were as-received from Sigma-Aldrich (Missouri, USA) with lot
number of E2645 and L8629, respectively. Alexa Fluor 488 EGF complex would be used directly
as-received from Invitrogen (California, USA); and CellTracker Red CMTPX were purchased from
Invitrogen as well. Pierce BCA protein assay kit was purchased from Thermo Scientific (Massachusetts,
USA). The anti-bodies for Alpha-Tubulin and EGFR (phosphor Tyr1068) were purchased from
GeneTex (Taiwan, ROC).
2-2 The modification of AFM tips and Sensor CM5 chips
EGF and PHA-E would be individually coated on the succinimide-modified silicon nitride
cantilever tips. The process was briefly described as follows. The tips were immersed in the solution of
EGF and PHA-E with concentration of 1 mg/ml, respectively, for 24 hours at 4°C and then washed
phosphate buffer solution (PBS). Finally, the residual succinimide ester was deactivated by
ethanolamine for 30 minutes at 4°C. The modified tips were stored in PBS at 4°C for later use.
EGFR was immobilized to the Sensor CM5 chips by amine coupling method. The chips were
cleaned with HBS-EP buffer (0.01 M HEPES, pH 7.4; 0.15 M NaCl, 0.005% v/v Surfactant P20) and
then soaked in EDC/NHS solution for activation overnight at 4°C. The EGFR were immobilized to the
surface-activated chips at 4°C. The residual N-hydroxysuccinimide ester was deactivated by
ethanolamine for 30 minutes at 4°C. The EGFR-immobilized chips were stored in HBS-EP buffer at
4°C for later experiments.
2-3 Adhesion force measurements
The topography and microstructure of the chip was obtained by MFP-3D-BIO AFM (Asylum
Research, California, USA) with tapping mode. The binding force between the test sample and EGFR
was measured on AFM by contact mode in PBS at room temperature. The location of immobilized
EGFR on the chip was identified to allow tip moving on for later measurement. Stress-strain curve
(force-distance curve) was obtained by moving the surface-modified tip to the EGFR-immobilized
location, holding it on for 3 seconds to allow binding to occur and then retracting. All the
measurements were executed at the same loading rate. The spring constant of the cantilever used in the
study was 0.06 N/m. The approach/retract velocity was 4 μm/s. Curves showing non-specific binding
events were not analyzed.
6
Adhesion force is measured in an AFM by collecting a force curve, which is a plot of cantilever
deflection (d) converted from a position sensitive photo-diode (PSPD). This deflection distance, as
function of sample position along the z-axis, is then converted into a force value acting on the spring
constant (k) of tip cantilever and relationship to Hooke's law [31]:
F  k d
(1)
The spring constant is determined from the individual frequency resonances with its shape factor which
is expressed as:
k  2wfL
3
(2)
E
where w is the width of cantilever, f is the measured resonant frequency, L is the length of cantilever, ρ
is the density of cantilever material, and E is the elastic modulus (Young's modulus) of cantilever
material [32].
2-4 Binding affinity analysis
The binding affinity of EGF and PHA-E to EGFR was measured by using a Biacore 2000 SPR
system (Biacore Life Science, Uppsala, Sweden) at room temperature. EGFR (200 μg/ml) was diluted
in 10 mM sodium acetate (pH 4.5) and immobilized to Sensor CM5 chip by using the amine coupling
method. The immobilization was completed when 10,000 resonance units (RUs) of EGFR was
achieved on experimental flow channel. The chip without EGFR on the other flow channel was used as
control. PBS was used as the running buffer and 50 mM NaOH was used for regeneration of the chip
surface. For each concentration of EGF and PHA-E, the flow rate of association and dissociation were
both 5 μl/min and regeneration was 100 μl/min. The equilibrium dissociation constant (KD) was
obtained to evaluate the binding affinity by using the BIAEvaluation software (Biacore Life Science,
Uppsala, Sweden).
Binding affinity is measured in a SPR by using kinetic method to calculate the equilibrium
dissociation constant (KD) [33,34]. This model displays the simplest situation of an interaction between
ligand (A) and immobilized receptor (B). The binding reaction equation of A and B:
ka
A  B  AB
kd
(3)
where ka is the association rate constant and kd is the dissociation rate constant. And the rate of product
7
(AB) formation at time t is written as:
d AB
 k a AB   k d AB (4)
dt
After some reaction time t:
B  B0  AB (5)
where
B0
is the concentration of B at t = 0. And taking (5) into (4) gives:
d  AB
 ka  AB 0  AB  kd AB
dt
(6)
AB complexes at the surface is proportionally formed and called signal R. Therefore, Equation (6)
becomes:
dR
 kaC Rmax  R   kd R
dt
where
(7)
dR
is the rate of formation of surface associated complexes, C is the constant concentration of
dt
A and Rmax is the capacity of A bound to B at surface. And the integrated form of the rate equation:
Rt 

Cka Rmax
1  e Ck a  k d t 
Cka  kd

(8)
This integrated rate is described the association phase of the binding curve. And the rate of dissociation
of the formed complexes (AB) is written as:
dR
 kd R
dt
(9)
The integrated form of the rate equation:
Rt  R0e  k d t (10)
where Rt is the response at time t and R0 is the amplitude of the response. We use equation (8) and
equation (10) to fit independently the data for association and dissociation phases respectively that
predict binding kinetic parameters.
8
2-5 Cell culture
A-549 (human lung adenocarcinoma) cells from BCRC (Taiwan, ROC) were cultured in nutrient
mixture F-12 Ham Kaighn's modification (Ham's F-12K) medium supplemented with 10% fetal bovine
serum (FBS) and 1.5 g/L sodium bicarbonate. The cells were maintained under standard cell culture
conditions at 37°C and 5% CO2. Typically, adherent cells were harvested with trypsin-EDTA and
re-suspended in a serum-containing medium before use in the assays as described below.
2-6 EGF binding assay
Commercialized EGF binding assay purchased from Invitrogen was used to evaluate how the
PHA-E competes with EGF bind to EGFR of cell membrane by flow cytometry. EGF as-received from
commercial kit was conjugated with green fluorescent protein (GFP). The intensity of fluorescence
would be detected when EGF bound to EGFR. A-549 cells, one of cancer cells with EGFR
overexpression, were seeded at culture dishes and incubated overnight. The medium was then replaced
with serum-free medium. After cultured further 24 hours, cells were harvested, washed and
re-suspended in 1 ml PBS. Cells were incubated only with EGF or with EGF and PHA-E at same time
in an incubator (37°C) for 15 minutes. Fluorescence was measured with a flow cytometer to in terms of
how much EGF binds to EGFR under the condition of PHA-E appear.
2-7 Immunofluorescent staining
Immunofluorescent staining was used to further check how PHA-E to compete with EGF bind to
EGFR. EGF was conjugated with GFP and the red fluorescence was CellTracker reagents which could
label the cytoplasm. The green fluorescence would be detected when EGF bound to EGFR and the red
fluorescence allowed tracing the cell location. The process was briefly described as follows. A-549
cells were seeded on a chamber slides and incubated overnight. The medium was replaced with
CellTracker dye working solution and reacted for 30 minutes. The working solution was then washed
and replaced with fresh medium. After cultured for another 30 minutes, the medium was replaced with
serum-free medium. Cells were incubated in the medium with EGF; or incubated in the medium with
EGF and PHA-E. After stayed in the incubator for 30 minutes, cells were washed with PBS for several
times. The chamber slides were mounted on confocal microscope for examination.
2-8 Western blot analysis
When EGF binds to EGFR, it would cause to EGFR phosphorylation and induce the series of
cellular reactions. In the study, Western blot analysis was used to detect EGFR phosphorylation as
9
follows. A-549 cells were seeded in petri dish and incubated overnight. The medium was replaced with
serum-free medium. After cultured for 24 hours, the medium was replaced with the medium containing
EGF. Another group was to replace the medium with EGF and PHA-E addition. After cultured for 45
minutes, they were washed with PBS. The cells were then directly solubilized in a lysis buffer
containing a protease inhibitor cocktail for 5 minutes on ice. The cells were then scraped and the lysate
was collected in an eppendorf tube. The lysate was cleared by centrifugation at 12,000 g for 15 minutes
at 4°C, and the protein concentration in the supernatant was determined by the Pierce BCA protein
assay. For Western blotting, equal amounts of proteins were resolved over 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF)
membrane. The non-specific sites on blots were blocked by incubating the membrane in a blocking
buffer (5% nonfat dry milk/TBST, pH 7.4) for 30 minutes at room temperature. Incubation was
performed with an appropriate monoclonal primary antibody in TBST overnight at 4°C, and then
incubated with a horseradish peroxidase (HRP) conjugated secondary antibody for 30 minutes at room
temperature. Immuno-reactive bands were visualized using an enhanced chemo-luminescence (ECL)
system.
2-9 Statistical analysis
Numerical data were presented as mean  standard deviation. Statistical differences among samples
were performed with Student's t-test. Statistical significance was considered at a probability P < 0.05
3. Results
3-1 The measurement of adhesion force by AFM
In this study, AFM was used to indirectly measure the adhesion force between EGF and EGFR. The
tip immobilized with EGF was mounted on the cantilever of AFM and scanned on Sensor CM5 chips to
trace the location where the EGFR immobilized on. Figure 1 showed AFM images of chip surface with
EGFR and without EGFR immobilization. The chip without EGFR immobilization showed a relatively
smooth surface (Figure 1 (a)) than that with EGFR immobilization (Figure 1 (b)). The AFM images
could be processed by the IGOR Pro MFP-3D software (Asylum Research) as shown in Figure 1 (c)
and (d). The spikes as high as 25 nm could be observed on the EGFR-immobilized chip (Figure 1 (d))
that would be indicated as the locations of EGFR where the EGF-immobilized tip would move to
measure adhesion force between EGF and EGFR for later interaction. The adhesion force between EGF
10
and EGFR were calculated and sorted by histogram and fitted with a single Gaussian curve to estimate
the adhesion force (Figure 2 (a)). The Guassian peak of histogram was at 158.624.30 pN. The average
adhesion force between EGF and EGFR was 183.8686.93 pN with n = 517 (Table 1). The same
procedure could be used to detect the adhesion force between PHA-E and EGFR. As shown in Figure 2
(b), the Gaussian peak of histogram was at 181.014.90 pN for PHA-E interaction with EGFR and the
average adhesion force was calculated as 207.1474.42 pN with n= 517 (Table 1). From the study, we
could tell that the adhesion force between PHA-E and EGFR was much higher than that of EGF and
EGFR (as Table 1); that could interpret as PHA-E has higher affinity to EGFR than EGF.
3-2 The evaluation of binding affinity by SPR
SPR was used to evaluate the binding affinity between EGF and EGFR by flowing ligand (EGF)
over EGFR-immobilized Sensor CM5 chip. In the study, different concentrations of EGF were injected
to the flow-channel and then flew over the EGFR-immobilized Sensor M5 chip. The binding affinity
between EGF and EGFR could be in terms of the dissociation constant (KD); which could be calculated
by using the BIAevaluation software (Biacore Life Science). As shown in Figure 3 (a), the KD value
between EGF and EGFR was about 7.3×10-82.7×10-8 as shown in Table 2. The binding affinity
between PHA-E and EGFR could be obtained from the same process as previous description (Figure 3
(b)). The KD value between PHA-E and EGFR was calculated as 2.4×10-91.4×10-9 (Table 2); that was
much lower than that of KD value between EGF and EGFR. The study provided one more piece of
evidence that the affinity of PHA-E to EGFR was higher than that of EGF to EGFR.
3-3 The competition of PHA-E and EGF bind to EGFR
EGF could effectively bind to EGFR and induce the downstream signaling pathways that would
be resulted in cell proliferation and survival of cancer. If targeting product of EGFR like PHA-E which
could compete with EGF to bind to EGFR on the surface of A-549 cells, the activity of receptor would
be inhibited that induce A-549 cancer cells apoptosis. It was therefore very important to investigate the
competition between PHA-E and EGF to EGFR of A-549 cells. EGF was conjugated with GFP as
received from commercialized product (EGF-GFP). The percentage of EGF-GFP binding to EGFR was
detected by flow cytometer. The experiment was divided into 7 groups: the untreated group was A-549
cells without any treatment, the group of EGF100 was A-549 cells treated with 100 ng/ml of EGF, the
group of EGF100+PHA-E8 was A-549 cells treated with 100 ng/ml EGF and 8 µg/ml PHA-E, the
groups of EGF100+PHA-E16, EGF100+PHA-E32, EGF100+PHA-E64, and EGF100+PHA-E128 was
11
A-549 cells treated with 100 ng/ml EGF and 16 µg/ml PHA-E, 32 µg/ml PHA-E, 64 µg/ml PHA-E,
and 128 µg/ml PHA-E, respectively. As shown in Figure 4, the fluorescence intensity was gradually
decreasing with the concentration of PHA-E; where the GFP peak was shifting to left side once the
treated concentration of PHA-E increased. The binding rate of EGF to EGFR were dropped as
99.7%0.1%, 99.4%0.2%, 97.7%0.7%, 94.3%1.3%, 84.8%3.4%, and 75.4%4.2%, when A-549
cells treated with 100 ng/ml EGF, 100 ng/ml EGF and 8 µg/ml PHA-E, 16 µg/ml PHA-E, 32 µg/ml
PHA-E, 64 µg/ml PHA-E, and 128 µg/ml PHA-E, respectively (Table 3). The results could prove that
the PHA-E could compete with EGF to bind to the EGFR effectively.
The study could be further proved by confocal microscope as shown in Figure 5. The green
fluorescence was the EGF bind to EGFR of A-549 cells and the red color was the cell tracker. We could
see that the green fluorescence was getting less and even no green color when examined at merged
picture with bright field (the far right column of Figure 5) once treated concentration of PHA-E up to
64 µg/ml. PHA-E treatment caused a dose-dependent decrease of the green fluorescence intensity.
3-4 The effect of competition between PHA-E and EGF on EGFR phosphorylation
EGF binding could activate EGFR to induce auto-phosphorylation of C-terminal tyrosine residues,
resulting in opening signaling pathway. If targeting product of EGFR like PHA-E which could not only
compete with EGF to bind to EGFR on A-549 cells, but also inhibit EGFR phosphorylation, the
downstream signaling pathways would be inhibited that induce A-549 cancer cells apoptosis. This
study was going to understand whether PHA-E could inhibit EGFR phosphorylation by reducing EGF
binding. The experiment was divided into 8 groups: the untreated group was the A-549 cells without
any treatment, the group of EGF50 was A-549 cells treated with 50 ng/ml of EGF, the group of
EGF50+PHA-E1 was treated with 50 ng/ml EGF and 1 µg/ml PHA-E, the groups of EGF-50+PHA-E2,
EGF50+PHA-E4, EGF50+PHA-E8, EGF50+PHA-E16, and EGF50+PHA-E32 was A-549 cells treated
with 50 ng/ml EGF and 2 µg/ml PHA-E, 4 µg/ml PHA-E, 8 µg/ml PHA-E, 16 µg/ml PHA-E, and 32
µg/ml PHA-E, respectively. As shown in Figure 6 (a), the expression of pEGFR(1068) was decrease
with increase of PHA-E concentration; where the α-tubulin was used as the internal standard in
Western blot. The results could be in terms of the bar chart as in Figure 6 (b). PHA-E treatment resulted
in a dose-dependent decrease of EGFR phosphorylation.
4. Discussion
12
NSCLC is the most common type of all lung cancers. Surgery is the standard treatment for
early-stage NSCLC; however, the majority of patients diagnosed at advanced stage are unsuitable for
surgical resection or extensive mediastinal lymphadenopathy [35,36]. Platinum-based chemotherapy
and/or thoracic radiotherapy is being the current standard of care for patients with unresectable
advanced NSCLC [37,38]. Unfortunately, despite these therapies, prognosis is poor, with the 5-year
survival rate of only about 16% for all stages [39]. Therefore, scientists started to look for other
therapeutic strategies. Among those, anti-EGFR or EGFR inhibitor was one of the most extensive
studies.
In recent years, raising understanding of the molecular biology and mechanisms of tumors has led
to the development of target therapy which improved the lack of target selectivity and toxicity profile
of traditional cancer treatments [40,41]. EGFR has been identified as an important therapeutic target in
the treatment of NSCLC because its high levels results in growth and survival of cancer [42,43]. EGFR
following pathways are often activated in cancer owing to the binding of ligands such as EGF [44,45].
Lots of EGFR inhibitors have been developed for the therapeutic purpose. However, most of the
inhibitors were generally induced serious side effects. PHA-E has been used to induce cell apoptosis
and cytotoxicity by blocking EGFR pathway on lung cancer cells [21], but it had not been thoroughly
explored how tightly bind to EGFR. As known, anyone of new EGFR inhibitor would face the
challenge of EGF competition; so that it is necessary to understand the adhesion force and binding
affinity between EGFR and PHA-E.
Adhesion force is measured in an AFM by collecting a force curve. The adhesion force is
characterized as the maximum force needed to begin separation of the ligand-receptor partners after
contact. Therefore, the binding between ligands and receptors can be identified by the increase in the
volume of the force [46]. In this study, we individually coated EGF and PHA-E on tips and
immobilized EGFR on chip to determine the adhesion force between ligands and receptors by AFM
(Figure 1). Our research suggests that there was higher adhesion force between PHA-E and EGFR than
that of EGF and EGFR as shown in Figure 2 and Table 1.
And then we used kinetic method to calculate the binding affinity by SPR [47]. We flowed different
concentrations of EGF and PHA-E by SPR and then calculated the equilibrium dissociation constant
(KD) by using the BIAEvaluation software (Figure 3). Our results suggest that EGF bound to EGFR
with higher KD than PHA-E (Table 2). There is an inverse relationship between KD and affinity, so the
13
binding affinity of PHA-E to EGFR was higher than EGF. Though PHA-E is provided with high
adhesion force and binding affinity to EGFR, it’s important to confirm whether PHA-E could compete
against EGF binding and then lead to inhibit EGFR phosphorylation.
Anti-EGFR mAbs have been developed not only has the ability to target but also can inhibit EGF
binding and receptor tyrosine phosphorylation [48,49]. The extracellular regions of EGFR are
composed of four subdomains. Ligand (EGF) binding stabilizes a domain rearrangement that domain
II/IV contact is broken and domains I and II rotate as a pair to bring domains I and III into proximity
and allow them to bind ligand simultaneously [50]. Our research demonstrated that EGF binding was
decrease when the dose of PHA-E was increase as shown in Figure 4 and Table 3. The results of
immunofluorescent staining showed the same trend of EGF binding inhibited by PHA-E (Figure 5).
Finally, we check whether PHA-E could enough to inhibit EGFR phosphorylation by blocking EGF
binding. Treatment of A-549 cells with PHA-E resulted in a dose-dependent decrease in the specific
phosphorylation sites of EGFR (Figure 6). PHA-E may inhibit EGF binding to EGFR through direct
competition with EGF by interacting with the EGF binding sites on the EGFR or indirectly blocking
the ligand binding by fixing a receptor conformation that reduce EGF binding affinity.
5. Conclusion
From the studies, we could conclude that PHA-E had better affinity to EGFR than that of the EGF.
The study could be proved by AFM, SPR, flow cytometer, confocal microscope and Western blot. The
interaction between PHA-E and EGFR could block EGF binding and then inhibit phosphorylation of
EGFR on lung cancer cells. PHA-E could be developed into a new therapeutic molecule for lung
cancer treatment and could be immobilized on the drug carrier as a targeting molecule to guide
therapeutic particles to the tumor site.
14
List of Abbreviations
AFM: atomic force microscope
EGF: epidermal growth factor
EGFR: Epidermal growth factor receptor
GFP: green fluorescent protein
KD: equilibrium dissociation constant
NSCLC: non-small-cell lung cancer
PHA-E: phytohemagglutinin erythroagglutinating
SPR: surface plasmon resonance
15
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Figure captions
Figure 1 The AFM pictures of Sensor CM5 chip surface immobilized (a) without EGFR and (b) with
EGFR. After images processed by the IGOR Pro MFP-3D, the clear pictures could be obtained as (c)
without EGFR immobilization and (d) with EGFR immobilization. The three-dimensional view with a
pseudocolor scale was ranged from low (purple) to high (red).
Figure 2 Adhesion force was described by fitting Gaussian curve indicated as red line; where (a) was
the EGF to EGFR and (b) was PHA-E to EGFR. (n = 517)
Figure 3 SPR was used (a) to evaluate the binding affinity between EGF and EGFR by flowing EGF
over EGFR-immobilized Sensor CM5 chip; and (b) to evaluate the binding affinity between PHA-E
and EGFR by flowing PHA-E over EGFR-immobilized chip.
Figure 4 The flow cytometer was to use to further check whether PHA-E could compete with EGF to
bind to EGFR on A-549 cells. The experiment was divided into 7 groups: the untreated group was
A-549 cells without any treatment, the group of EGF100 was A-549 cells treated with 100 ng/ml of
EGF, the group of EGF100+PHA-E8 was A-549 cells treated with 100 ng/ml EGF and 8 µg/ml PHA-E,
the groups of EGF100+PHA-E16, EGF100+PHA-E32, EGF100+PHA-E64, and EGF100+PHA-E128,
was A-549 cells treated with 100 ng/ml EGF and 16 µg/ml PHA-E, 32 µg/ml PHA-E, 64 µg/ml PHA-E,
and 128 µg/ml PHA-E, respectively.
Figure 5 Immunofluorescent images showed the competing binding of EGF and PHA-E to EGFR on
A-549 cells. A-549 cells were treated with EGF (500 ng/ml) and PHA-E (in different concentrations)
for 30 min and then examined under confocal microscope. The green fluorescence was the EGF
conjugated with green fluorescent protein (GFP) and the red fluorescence was the cell tracker to label
the cytoplasm.
Figure 6 The effect of PHA-E on EGFR phosphorylation was checked by Western blot, where (a) the
expressions of EGFR phosphorylation; and (b) the quantization of Western blot images. The α-tubulin
was used as internal standard. The experiment was divided into 8 groups: the untreated group was the
A-549 cells without any treatment, the group of EGF50 was A-549 cells treated with 50 ng/ml of EGF,
the group of EGF50+PHA-E1 was treated with 50 ng/ml EGF and 1 µg/ml PHA-E, the groups of
EGF-50+PHA-E2, EGF50+PHA-E4, EGF50+PHA-E8, EGF50+PHA-E16, and EGF50+PHA-E32 was
A-549 cells treated with 50 ng/ml EGF and 2 µg/ml PHA-E, 4 µg/ml PHA-E, 8 µg/ml PHA-E, 16
µg/ml PHA-E, and 32 µg/ml PHA-E, respectively. (*P < 0.05 and **P < 0.01 (n = 3) vs. the group of
EGF50)
20
(a)
(c)
(b)
(d)
Figure 1
21
(a)
(b)
Figure 2
22
(a)
(b)
Figure 3
23
Figure 4
24
Figure 5
25
(a)
(b)
Figure 6
26
Table 1 Summary of average adhesion force of EGF and PHA-E to EGFR based on Figure 2 (n = 517)a
Ligands
EGF
PHA-E
Adhesion force to EGFR (pN)
183.65±86.93
207.14±74.42 **
a
Data are presented as mean ± SD (** P＜0.01)
Table 2 Summary of the equilibrium dissociation constant (KD) of EGF and PHA-A to EGFR based on
Figure 3 (n = 3)a
Ligands
EGF
PHA-E
KD (M)
7.3×10-8±2.7×10-8
2.4×10-9±1.4×10-9 **
a
Data are presented as mean ± SD (** P＜0.01)
Table 3 Binding rate of EGF to EGFR in the different concentrations of PHA-E presence based on
Figure 4 (n = 3)a
Group
Untreated
EGF100
Binding rate of
EGF to EGFR
0.1%
**
99.7%±0.1%
a
EGF100+
PHA-E8
99.4%±0.2%
*
EGF100+
PHA-E16
97.7%±0.7%
**
EGF100+
PHA-E32
94.3%±1.3%
**
EGF100+
PHA-E64
84.8%±3.4%
**
Data are presented as mean ± SD (* P < 0.05 , ** P < 0.01 vs. the group of EGF100)
27
EGF100+
PHA-E128
75.4%±4.2%
**